Purified ferrimycin and process for obtaining same

ABSTRACT

The antibiotic ferrimycin A, its components ferrimycin A1 and ferrimycin A2 and the corresponding iron-free compounds desferrimycin A, desferrimycin A1 and desferrimycin A2, and also pharmaceutical preparations which contain these products, and a process for the manufacture of these substances and mixtures containing them. The antibiotic ferrimycin belongs to the sideramycins, a class of iron-containing or iron-binding antibiotics, to which also the antibiotics grisein, albomycin and A 1787 belong. The sideramycins are characterized by their antibiotic effect being antagonized by the ferrioxamines; see Bickel et al., Experientia 16 (1960) 129.

United States Patent [191 Gaeumann, deceased et al.

[4 11 Dec 3, 1974 PURIFIED FERRIMYCIN AND PROCESS FOR OBTAINING SAME[75] Inventors: Ernst Gaeumann, deceased, late of Zurich, Switzerland;Tino Gaeuman,

legal representaitve, Mont Sur Lausanne; Vladimir Prelog, Zurich; ErnstVischer, Basel; Hans Bickel, Binningen, all of Switzerland [73]Assignee: Ciba-Geigg Corporation, Ardsley,

[22] Filed: Oct. 19, 1966 21 Appl. No.: 626,650

Related US. Application Data .[63] Continuation-impart of Ser. No.245,349, Dec. 11,

1962, abandoned',,which is a continuation-impart of Ser. No. 32,294, May27, 1960, abandoned, which is a continuation'in-part of Ser. No.749,616, July 21, 1958, abandoned.

so Foreign Application Priority Data July 26, 1957 Switzerland 48868/57July 8, 1958 Switzerland... 61491/58 May 29, 1959 Switzerland.....73755/59 Mar, l8, 1960 Switzerland 3062/60 [52] U.S. C1. 424/118, 195/80[51] Int. Cl A6lk 21/00 [58] Field of Search 424/118; 195/80 [5 1References Cited UNITED STATES PATENTS 3,033,760 5/1962 Gauemann 195/80OTHER PUBLICATIONS Antibiotics & Chemotherapy, 12, 3445 1962Experientia, 16, (1960) article commencing page 128.

Helv. Chim. Acta, 43, (1960), pg. 901

Arch. Mikrobiol, 36, (1960) article commencing page 325.

Helv. Chim. Acta, 43, (1960), article commencing page 2105.

Arch. Mikrobiol, 38, (1961) page 326-338;

Primary Examiner-Jerome D. Goldberg Attorney, Agent, or FirmJoseph G.Kolodny 57 ABSTRACT 4 Claims, 8 Drawing Figures I PATENIEL; DEC 31974SHEET 2 BF 8 v cor PATENTEL EEC 74 SPEET 3 0F 8 PAIENHL WI 31974 T acw 8com com camp

ucm ooou coma coon

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as on in? 2 5 g PURIFHED ,FERRIMYCIN AND PROCESS FOR OBTAINING SAME Thisis a continuation-in-part of our application Ser. No. 245,349, filedDec. 11, 1962 (now abandoned), which is itself a continuation-in-part ofour application Ser.No. 32,294, filed May27, 1960 (now abandoned), whichis itself a continuation-in-part of application Ser. No. 749,616, filedJuly 21, 1958 (now abandoned), by Vladimir Prelog et al.

The invention relates to a new watersoluble antibiotic which we formerlydesignated as A 9578 or as pilosomycin and is now called ferrimycin A,its components ferrimycin A and ferrimycin A and thecorrespondingiron-free compounds desferrimycin A, desferrimycin A anddesferrimycin A and also pharmaceutical preparations which contain theseproducts, and a process for the manufacture of these substances andmixtures containing them.

The antibiotic ferrimycin belongs to the sideramycins, a class ofiron-bindingantibiotics, to which also the antibiotics grisein,albomycin and A 1787 belong. The sideramycins are characterized by theirantiobiotic effect being antagonized by the ferrioxamines; see

Bickel et al., Experientia 16 (1960) 129.

Ferrimycin and its components are red-brown, basic substances which arereadily soluble in acids and strongly polar solvents, such as water,methanol, dimethylformamide, glycol,.methyl cellolsolve. They alsodissolve in benzyl alcohol, in phenols or in mixtures of phenols andlipoid solvents. They consist of hydrocarbon, hydrogen, oxygen andnitrogen, and also contain iron or are capable of bindingiron.-Ferrimycinis a mixture of closely related compounds which consistof two main components, ferrimycin A and ferrimycin B. Ferrimycin A canbe separated into two components, ferri mycin A l and ferrimycin A 2.The properties of these various components are described below.

It has not hitherto been possible to obtain ferrimycin or theabove-mentioned components in a pure form.

The special difficulty in purifying the ferrimycinsarises principallyfrom the fact that they are present in the culture filtrate in verysmall proportion in addition to large amounts of inactive substancesfrom the nutrient solution and fermentation products having very similarphysical-chemical properties. The culture filtrate contains on anaverage for every one part of ferrimycin 10,000l5,000 parts by weight ofinactive substances which are hydrophilic, like the ferrimycins. Thedifficulty of enriching and purifying the ferrimycins alsov arises fromtheir instability in a wide range of pH values. The separation of theantibiotic into its individual components is also rendered difficultowing to the fact that these components differ from one another onlyslightly in their R values even in chromatographically favorable solventsystems.

Antibiotic ferrimycin is obtained by the culture of a new strain ofactinomycetes, of the species Streptomyces griseflavus, which has beenisolated from a sample of soil collected in Boston, Mass, and which iskept in our laboratories, and also in the Eidg. Technische Hochschule,Institut fur spezielle Botanik under the designation A 9578 and in theU.S. Department of Agriculture, Agricultural Research Service, NorthernUtilization Research and Development Division, Peoria, 111., under thedesignation NRRL 2717.

Streptomyces griseoflavus NRRL 2717 belongs to the species S treptomycesgriseoflavus (Krainsky) Waksman and Henrici, and differs from othermembers of this species in that it forms a new water-soluble antibiotic.Hitherto only one strain of Streptomyc es griseoflavus is known whichproduces an antibiotic. This is the so-called griseoflavine, whichdiffers from the new antibiotic ferrimycin in its solubility in organicsolvents. The air mycelium of Streptomyces griseoflavus NRRL 2717 is ashgrey. The spore carriers are branched and form copious spirals generallyhaving 2-5 turns. The spores themselves have a size of 1.0 to 1.3g. X0.7 to

0.9 1., and have at their surface spines about 0.2 y inlength whicharepointed and only slightly widened at their base. Their growth isrelatively little dependent on temperature, so that the mould developswell at 18C and also at C, although the optimum temperature is between25C and 32C.

For the purpose of identification there is described below the growth ofStreptomyces griseoflavus NRRL 2717 on various nutrient media. Thenutrient media numbers l-7 and also 10 are prepared as described by W.Lindenbein, Arch. Mikrobiol. 17, page 361 (1952).

Growth cloud-like colorless, no air mycellium. Sediment, reddish flocks,Well developed pellicle having cloud-like white-grey air mycellium.Substraturn brown-yellow to deep yellow. Growth initially point-like,wrinkled after 12 days and pale yellow. Growth initially point-like,later cloudy and yellowish.

' Air mycellium velvety,

initially brownish grey, later ash grey. substratum yellow. I Growthcloud-like, white- I yellow, later deep yellow, Air mycellium scanty, 1covered with flour-likedust, floury white. substratum pale brown.

1 Growth very scanty,

liquefaction very slow (0.5 cm after 30 days). Growth cloud-like,milkwhite, no air mycellium. Hydrolysis 1 cm after 5 days.

Growth initially point-like, pale yellow, later pimply. Air mycelliumvelvety, white yellow to ashgrey. substratum colored brownish 1)Synthetic agar: 2) Synthetic solution:

3) Glucose-agar:

4) Glucose-asparagineagar:

5) Calcium malate-agar:

6) Gelatine stab culture 7). Starch plate:

8) Potatoes:

' grey. 9) Carrots: Growth very scanty. Air

mycellium scanty. Substratum not colored. Ring growth and surface skincolorless. Air mycellium white-grey, hydrolysis slow withoutco-agulation, litmus blue.

Negative.

l0) Litmus milk:

Tyrosinase reaction:

the use of Streptomyces griseoflavus NRRL 27l7 or other strainscorresponding to the description thereof, but also includes the use ofvariants such, for example, as are obtained by selection or mutation,especially under the action of ultra-violet rays or X-rays or nitrogenmustard oils.

In order to produce antibiotic ferrimycin A or its components a strainof Streptomycetes having the properties of 'Streptomyces griseoflavusNRRL 2717 is incubated aerobically, for example, in an aqueous nutrientsolution containing a source of carbon and of nitrogen and inorganicsalts until the solution exhibits a substantial antibacterial action,and the antibiotic ferrimycin is then isolated.

As source of carbon there may be used, for example, glucose, saccharose,lactose, mannitol, starches or glycerine. As nitrogenous nutrientsubstances and, if desired, growth promoting substances there may bementioned amino-acids, peptides and proteins, and also their degradationproducts such as peptone or tryptone, and also meat extracts,water-soluble constituents of cereal grains, such as maize or wheat, orof the distillation residues from the manufacture of alcohol, or yeast,or beans, especially soya bean plants, or seeds,

. for example, those of cotton plants etc., and also ammonium salts andnitrates. Among other inorganic salts the nutrient solution may containchlorides, carbonates, sulfates of alkali metals, alkaline earth metals,magnesium, iron, zinc and manganese.

The incubation is carried out aerobically, for example, in a quiescentsurface culture or advantageously submerged while agitating or stirringwith air or oxygen in agitated flasks or known fermenters. The.temperature may be within the range of 18C to 40C. The nutrient solutiongenerally exhibits a substantial antibacterial action after 1 to 5 days.

. In order to isolate antibiotic ferrimycin the following methods may beused: The mycelium is separated from the culture filtrate, and thegreater part of the antibiotic is-found in the culture filtrate.However, considerable quantities of the antibiotic are adsorbed on themycelium. It is therefore of advantage to wash to mycelium well. Forthis purpose water or an aqueous organic solvent may be used, such as analcohol, for example, aqueous methanol. In order to recover theantibiotic from the culture filtrate and to purify it, various methodsmay be used which can be employed singly or in combination with oneanother. It is of advantage during these operations to'main'tain theculture solution at pH value within the range of 35.

1. For isolating the crude ferrimycin from the culture filtrate, andadsorption medium may be used, for example, an active carbon such asNorit, activated earths such as F ullers earth or Floridine(aluminummagnesium silicates) or a resin adsorbant such as Asmit (ameta-phenylenediamine-formaldehyde decolorizing resin). The adsorbate'is advantageously eluted with a mixture of water and an organic solventmiscible with wateror an aqueous acid, for example, a mixture of waterand methanol, water and pyridine, dilute acetic acid and methanol, orwater, methanol, glacial acetic acid and butanol. Especiallyadvantageous for the elution of a Norit-adsorbate is a mixture of 2parts by volume of water, 1 part by volume of methanol, 1 part by volumeof glacial acetic acid, and 2 parts by volume of butanol.

2. A second method for separating the antibiotic from the culturefiltrate is to adsorb the antibiotic on a cation-exchanger, for whichpurpose resins containing acid groups, such as Amberlite IRC-5O (acarboxylic acid type ion exchanger) are especially suitable. The lattercan be'used either in the acid form or in the sodium form, although amixture of both fonns in the volumetric ratio 1:2 is particularlyadvantageous. Elution is advantageously carried outwith a dilute acid,e.g. methanolic hydrochloric acid.

3. The basic antibiotic can also be precipitated directly from theculture filtrate, for example by reaction with an organic acid of thetype of picric acid. By treating a precipitate so obtained with a saltof an organic base, for example, with triethyl-ammonium sulfate, or

with a dilute acid, the antibiotic is obtained in the form of thecorresponding salt. These operations may be carried out either in anaqueous medium or in a solvent miscible with water, such as methanol oracetone. The conversion of the sparingly soluble salts into the readilysoluble salts of the antibiotic is carried out either by means ofmineral acids or by treatment with ion exchanging resins, e.g. AmberliteIRA-400 (a polystyrene resin containing quaternary ammonium groups).

4. The antibiotic can be concentrated by adding to an aqueous oralcoholic-aqueous solution of the salt of the antibiotic an excess of anorganic solvent miscible with water, such as acetone, dioxane etc.,whereby the salt is precipitated in solid form.

5. Another method of enriching the antibiotic consists in extracting anaqueous solution thereof with a solution of phenol in chloroform inwhich operation both the pH of the aqueous solution and the phenolcontent of the chloroformic solution can be "varied. As an example, in adistribution between a solution containing in 100 cc of chloroform 100grams of phenol and an aqueous phase having a pH of l to 6, nearly allof the antibiotic is in the organic phase, whereas, when a solution isused which contains only 33 grams of phenol in 100 cc of chloroform, itcan be extracted nearly creases as the phenol content of the organicphase in- 1 creases, and decreases as the pH of the aqueous phase falls.It thus being possible to chose anydesired distribution coefficient ofthe antibiotic in this system, a large proportion of inactive impuritiescan be eliminated. by combining a small number of distributingoperations. Y

6. There is still another method of enriching the antibiotic, namely,chromatography, such as adsorption chromatography on various materials,e.g. Norit' (activated carbon), alumina, magnesium silicates, silicagel, calcium sulfate, or distribution chromatography with cellulose,starch, silica gel, Celite (an infusorial earth) or the like as carriersubstances, or chromatography on ion exchangers, e.g.,' Dowex (asulfonated polystyrene), AmberlitelRC-SO (a carboxylic acid type cationexchanger) or the like. Good results have been obtained for example withdistribution chromatography on cellulose using the solvent system of 4parts by volume of butanol, 1 part by volume of glacial acetic acid, and5 parts by volume of water.

7. The antibiotic can also be enriched by the countercurrentdistribution according to Craig between two immiscible solvent phases.The following solvent systems have proved particularly successful:

a. secondary butanol 1/10 N-ammonium acetate buffer having a pH of 4.68.

b. 1 N-ammonium acetate buffer having a pH of 46-10% solution of phenolin chloroform. The distribution coefficient of the antibiotic in thesystem (b), and thus the location of the activity maximum in thedistribution can be changed as desired on the one hand by changing thepH of the buffer solution and on the other hand by changing the phenolcontent of the organic phase.

The following combination of the enriching methods described above givespreparations of considerable purity. From the culture filtrate, theantibiotic is adsorbed on the buffered ion exchanger Amberlite lRC-SOand eluted by means of methanolic hydrochloric acid. At a pH of 5, theeluates are concentrated under reduced pressure, an aqueous concentrateof the antibiotic being obtained in an amount of about 1/ 100 of thevolume of the nutrient solution. By method (5 above, the concentrate isdistributed several times between phenol-chloroform mixtures and aqueoussolutions of varying pH. Oh freeze-drying the resulting active solutionthere is obtained a preparation the specific activity of which is500-1000 times higher than that of the lyophilized culture filtrate. Aspecific embodiment of the present invention consists in furtherpurifying the crude ferrimycin as obtained according to the abovementioned methods by subjecting it to electrophoresis and- /orcountercurrent distribution with the use of benzyl alcohol and, ifdesired, chromatography.

The electrophoresis is carried out in the form of a high voltageelectrophoresis at 500 to 4,000 volts, in the form of zonalelectrophoresis, and especially countercurrent electrophoresis. V r

In the case of zonal electrophoresis in dilute acetic acid the activity.of ferrimycin A is increased to 7,000 to 8,000 times the activity of thelyophilized culture filtrate.

times) is obtained in the counter-current electrophoresis of ferrimycinA. in this method of separation the antibiotic is present as a locallyfixed cation, in that the tendency for movement by an electric field isexactly counteracted by means of an oppositely directed stream of theelectrolyte. Substances that undergo electrical movement in a differentmanner leave the apparatus at either one of the two ends, where theelectrodes are fixed.

For chromatography there are used as adsorbents preferably strong acidion-exchange resins, such as Dowex SO-WX (a sulfonated polystyrenecontaining 2 percent of divinylbenzene). As eluting agents there areadvantageously used basic buffer solutions of increasing concentrations,such as ammonium acetate buffers of pH value 4.6 in a molecularconcentration of 0.2 to

For countercurrent distribution there are used, for

example, the following systems:

Benzyl alcohol (60 parts by weight) methylisobutylketone (44 parts byweight) aqueous sodium chloride solution of 15% strength parts byweight) 0.0 1 N-hydrochloric acid (50 parts by weight); Benzyl alcohol(66 parts by weight) rne thy lisobutylketone (3 30 are ey Weight) '1aqueous sodium chloride solution of 5% strength (50 parts by weight)0.01N-hydroch1oric acid (50 parts by weight). Also suitable is thesystem benzyl alcohol (200 parts by volume)-n-butanol (100 parts byvolume) water (300 parts by volume) saturated, aqueous sodium chloridesolution parts by volume) N-hydrochloric acid (6 parts by volume). Bythe use of of these systems the active substance is divided into the twocomponents ferrimycin A and ferrimycin B.

The two componentsA and B ferrimycin are defined by paper-chromatographyby a direct comparison of their Rf values with the Rf values of aseries'of known antibiotics (2-11) in the systems A G. In the case ofsystem H the figures represent in cm the distance travelled by theantibiotics after 16 hours. The antibiotics are detectedautobiographically with Staphylococcus aureus or Bacillus subtilis.

Sy- 1a lb 2 3 4 5 6 7 8 9 10 11 stern A 0 0 0 0 0 0 0 0 0 0.92 0.07 0 B0.49 0.63 0.05 0.05 0.17 0.02 0.02 0.66 0.55 0.92 0.32 0.22 C 0.34 0.580.22 0 0.02 0.22 0.03 0.72 0.62 0.93 0.39 0.11 .D 0.05 0.15 0 0 0 0 0 XX 0.92 -0 E 0.32 0.32 0 0.10 0.22 0 0 X X- 0.86 0.12 F 0.47 0.47 0.220.14 0.12 0.04 0.05 0.49 0.42 0.91 0.43 0.36 G 0.74 0.74 0.07 t r 0.02 XX 0.94 0.61 v 0.69 11 2.7 7.6 0 0 0 0 0. (14.5) (8.8) 27 i l Xantibiotic distribution over the whole course position unsharp Awater-saturated butanol B'butanol-glacial acetic acid-water (421:5)(upper phase) C water-saturated butanol 2% paratoluene-sulfo acid Dwater-saturated butanol 2% piperidine E butanol-pyridine-water (614:3)

F ethanol 1.5% NaCl. Whalman No. 4 impregnated with 0.95 molar Ni So0.05 molar NaHSO G butanol-cthanol-water (1:112) H hutanol-butylacetate-glacial acetic acid-water (l0:3:1.3:14.3) (upper phase) 1::ferrimyein base A 1b ferrimycin base B 2 Streptomycin 3 Rislocetin A 4Ristocetin B 5 Neomycin B 6 Viomycin 7 Chlorotetracyclin 8Oxytetracyclin 9 Actinomycin l 10 Cycloscrin 1 1 Grisein During paperelectrophoresis in an 0.1 molar acetate buffer having a pH ,value of 4.6antibiotic ferrimycin migrates towards the cathode. The speed ofmigration is about half as great as that of Streptomycin.

For separation into the components A1 and A2 distribution chromatographyon cellulose in the system n-butanol-0.5N-a'cetic acid (1:1) isespecially suitable.

The resulting preparations have 10 to 20,000 times the acitivity of thelyophilized culture filtrate, whereas the products used as startingmaterial have only 100 to 1,000 times the activity of the lyophilizedculture fil-. trate. A ferrimycin A preparation purified in thedescribed manner, a strongly acidic ion exchanger being used in the lastpurification step, shows, in the form of the dihydrochloride, thefollowing chemical and physical properties:

Microanalysis:

C:48.65%, l-I:7.09%,

N:12.95%, Fe 4.56%,

CI: 6.10%, (C)CI-I 1.99% (Roth-Kuhn), Amino-N:

2.33% (Van Slyke). Titration:

pl(*,,, (Helv. 37,1872 (1954)):

Equivalent weight: 1106. Absorption spectrum:

A max:' 228 mu, E 282 max: 319 mu, E

28.2 max: 425 mu, E 22.6

Reduction value according to C. S. Hanes, Biochem. J.23, 99 (1929); 1.7ml l/lOO N-sodium'thiosulfate. Bound hydroxylamine according to T. Emeryand J. B. Neilands, Nature 184, 1632 (1959): 0.83 mol NH OH per atom Fe.

Ferrimycin A is an orange-yellow powder which dissolves very readily inwater, methanol and mixtures of phenol and chloroform, dissolves indimethylformamide, methylcellosolve, benzyl alcohol and glacial aceticacid, dissolves sparingly in ethanol and is practically insoluble inpyrridine, propanol, butanol and the usual organic solvents, especiallylipoid solvents. It can be precipitated from an aqueous solution bymeans of 1 picric acid, picrolonic acid and ammonium reineckate. Theorange-red aqueous solutions of the antibiotic change color reversiblythrough claret red to pale yelalso brought about with sodium hydroxidesolution. Neutral solutions do not react with potassium ferrocyanide,and acidified solutions yield colorations or precipitates of Prussianblue. The trivalent iron bound in The componentsA and A arecharacterized as follows:

A -dihydroch1oride: Elementary analysis (after drying for hours at 25C/1 0 mm over P O /KOI-I; average from three determinations) yields: C46.71%; H 6.80%; N 12.70%; C1 6.76%; Fe (color.) 5.33%; 0 (calc.21.70%). Titration in 80% methylcellosolve; equivalent weight: 1078; pK4.11; 7.92; 1 1.4. These values suggest the empirical formula C l-l O NFe, I-ICl, and the molecular weight 1051. Its ultraviolet spectrum showsmaxima at 229 my. (E 336), 319 mp. (E 37) and 425 mu (E 27.6). Itsinfrared spectrum.(in potassium bromide) is given in FIG. 2. It showsbands at 3.00; 3.44; 6.08; 6.31; 6.90; 7.35; 7.45; 7.95; 8.41; 9.00;10.25; 11.85; 13.20;/..

A -dihydrochloride: Elementary analysis (after drying) as above) gives:C =45.78%; H 6.77%; N 12.75%; C1 6.23%; Fe (color.) 5.29%; titration in80% methyl-cellosolve; molecular weight: 1086; pK 4.04; 7.91; 11.05.These values suggest the empirical formula C, H O N Fe, l-lclltsultraviolet spectrum shows maxima at 227 mp (E 332), 319 mp. (E 37) and425 mp. (B 32 25). Its infra-red spectrum (in potassium bromide) isshown in FIG. 3. It shows bands at the same positions as the spectrum ofcomponent A In FIG. 5 are shown the ultraviolet spectra (in water: c= 5X 10*) of purified ferrimycin A (Curve 1, Extinction E A), of thecomponent A (Curve 2, E A 0.1) and of the component A, (Curve 3, E =A0.2).

The ferrimycins can be definitely distinguished by paper chromatographyfrom Grisein (F. A. Kuehl, M. N. Bishop, L. Chaiet and K. Folkers, Am.Soc. 73, 1770 (1951)), Albomycin [G. F. Gause, Brit. Med. J. 1955, 1177]and A 1787 [H. Thrum, Naturwiss. 44, 561 (1957)]. In the systemn-butanol-glacial acetic acidwater 4:1:5 (see FIG. 4) these antibioticsare still almost at the starting point aftera running time of 10 hours.The test is carried out bio-autographically with Staphylococus aureus.

low on'the addition of mineral acid. Decoloration is 5 complex union isliberated when the pH value is de- 50 creased.

Ferric chloride causes a claret red coloration and ferric chloride pluspotassium ferricyanide give a blue coloration. The following tests arenegative: Molish, Anthron, Folin-Ciocalteu and Sakaguchi. Its ninhydrinreaction in a mixture of butanol and pyridine is only slightly positiveafter heating for a long time. Hydrolysis with 6N-hydrochloric acidyields a mixture of about 15 substances detectable by paperchromatography. Among these compounds the following can be identified:succinic acid, 1amino-Shydroxyaminmpentane, S-aminovaleric acid,cadaverine, ammonia, proline, a crystalline substance (C H O N.HCI) withmax 227 and 323 my. and ferric chloride.

The purified ferrimycin A and its components A and A respectively reactwith 8-oxyquinoline in solution in methanol with the separation of ablack colored crystalline precipitate of iron oxyquinoline. Theiron-free ferrimycin A can be isolated from the solution as a yellowishpowder. It is also obtained when ferrimycin A is treated with alkali orwith strong mineralic acids. Desferrimycin A possesses one half of theactivity in vitro of the starting material, and exhibits, apart from theabsence of the iron band at 425 mu, the same absorption spectrum asferrimycin A. By the addition of ferric chloride its specific activityin vitro is doubled and the found in desferrioxamine B occur also indesferrimycin The purified base A can be split up by chromatography intocomponents A and A As an adsorbent it is of advantage to use cellulose,and as the flow agent tertiary butanol/0.001 N-hydrochloricacid/saturated aqueous NaCl solution 2:12].

A Additionally desferrimycin A contains a phenolic constituent. Uponhydrolysis of desferrimycin A with 6-n. hydrochloric acid at roomtemperature there is obtained this phenolic compound bound to one 1amino-Shydroxylamino-pentane residue, besides two moles ofN-(5-hydroxylaminopentyl)-succinimide hydrochloride and one mol ofacetic acid. The formula of the phenolic hydrolysis product is C H O N 3l'lCl. It yields, on hydrolysis with l-n. hydrochloric acid at l C,1-amino-5-hydroxylamino-pentanedihydrochloride, ammonium chloride and aphenolic aminocarboxylic acid hydrochloride of the formula c gHzlogNg,l-lCl. The latter yields, on hydrolysis with 6-n. hydrochloric acid at 110C, the crystalline hydrochloride of 3-amino-5-hydroxybenzoic acid, C HO N, l-lCl. From the different hydrolysis products and their spectra thefollowing formula can be deduced for ferrimycin A -dihydrochloride:

out

ular weight 998 for the dihydrochloride of desferrimycin A DesferrimycinA shows in the UV-spectrum in ethanol maxima at )t 210 (log 5 4.57); 233(log e =4.38) and 322 (log 6 =3.22') mu. In 0.01 N alcoholic sodiumhydroxide there are maxima at 230 my. (log 6 2.61) and 335 mu (10g 1'm1.54) and a weak shoulder at 245 my. (log e q 2.48). The IR- spectrum inliquid petrolatum shows bands at 845; 930 (shoulder); 975; 1002; 1022;1057; 1125; 1161; 1195; 1225; 1259; 1305 (shoulder); 1378; 1466; 1550;1612; 1677 (shoulder); 1715 cm (cf. FIG. 7).

The NMR-spectrum in trifluoracetic acid is given in FIG. 8.

The antibiotic ferrimycin A and its components ferrimycin A andferrimycin A and the corresponding iron-free compounds can easily beobtained in the form of the free base from a salt thereof, for example,from the sulfate, for example, by reaction in an aqueous medium withbarium hydroxide, neutralization of the ex cess of baryta with carbondioxide, separation of the precipitate of barium carbonate and bariumsulfate, and isolation of the free base by freeze drying. A simplemethod of producing the base from its salts is to use a strongly basicanion-exchanger, for example, the OH- form of the product known incommerce as Dowex-2 (a polystyrene resin containingdimethylethanolamine).

One of the most remarkable properties of ferrimycin A and components isits pronounced stability minimum between pH values of 7 and 1 1.Solutions of the antibiotic at 20C and at a pH value of 8-10 lose theiractivity in the course of 48 hours, whereas at the same tempera ture andat pH values of 1-5 they remain fully and at pH values 6-7 or greaterthan 111 partially active.

Salts of antibiotic ferrimycin A and its components and thecorresponding iron-free compounds can be obtained from the knowninorganic and organic acids, for example, hydrochloric acid, sulfuricacids and phosphoric acids, acetic acid, propionic acid, valeric acid,palmitic acid, or oleic acid, succinic acid, citric acid, mandelic acid,pantothenic acid, glutamic acid or other amino acids. They are neutralor acid salts. They can be prepared by the action of the correspondingacids on the free base or by the double decomposition of salts, forexample, of ferrimycin-sulfate with calcium pantothenate.

Antibiotic ferrimycin A, ferrimycin A and ferrimycin A and thecorresponding iron-free compounds have a very high antibiotic activityagainst various test organisms. In the so-called agar cross streak testthey are active against the following test organisms: Micrococcus.pyogenes var. aureus, Streptococcus pyogenes, Streptococcus vin'dans,Streptococcus faecalis, Corynebacterium diphteriae, Escherichia coli,Bacillus megatherium and Bacillus subtilis.

The bacteriostatic activity of the ferrimycins, for in stanceagainstStaphylococcus aureus, can be used as a measure for the activityof preparations of different degrees'of purity. One unit is defined asthe activity which in 20 ml of meat extract agar (contained in aPetri-plate of 9 cm diameter) inoculated' with about'200 germs of Staph.aureus ATCC 6538 and incubated for 24 hours at 37C suppresses 50% of thegrowth. The culture filtrate has an activity of 2,0005,000 units/m1. Theactivity of the purest preparations of ferrimycin A was found to be900,000 units/mg; that of ferrimycin A 1,000,000 1 100,000 units/mg andthat of ferrimycin A 1,100,000 r 100,000 units/mg.

1n the following Table are given the activities in vitro of purifiedferrimycin products. There are given in mil- Strain Ferrimycin AFerrimycin A Ferrimycin A,

Staph. aur. 26 27 26.5 Strepto. face. 8 9 9.5 Esch. coll 10.5 10.5 11Shigella sonnei 13.5 14 l 14 Klebs.typ.A 23* 22.5" 22.5 Past.pestis 1212 12 Bac.megatherium 20 20 20 Ustilaga sphaemgena 28 Uslilagoscabt'osae 24 Cloudy zones of inhibition Table I Infection with Staph.aureus Strept. haemol. Pneumococc. lll

s.c. p.o. s.c. p.o. s.e. p.o.

Ferrimycin A, 0.1 5 0.1 3.3 0.33 3.3 1- Penicillin-G 1 3.3 5 3.3Erythromycin 5 100' 50 or corresponding iron-free compound Table I:Doses (in mg/kg) of different antibiotics which cure 75l00%-of theinfected animals with live subcutaneous or oral doses within 30 hours(observation period at least 10 days)v lnthepaper disc fest the diameters of'the zones of i'n From the above Table it is clear that ofthTtlir eeantibiotics investigated the ferrimycins or desferrimycins respectivelyare the most effective against Staphylococcus aureus and Streptococcushaemolyticus when administered subcutaneously, being quantitatively 10to 50 times superior to penicillin and 50 to 100 times superior toerythromycin. When administered orally, the

new antibiotics have about the same effect as penicillin, but aredefinitely superior to erythromycin.

Even when administered onlyonce, the ferrimycins or desferrimycinsrespectively show a superior effect.

if rimes Staph. aureus Strept. haemol Pneumococc. lll s.c. p.o. s.c.p.o. s.c. p.o.

Ferrimycin A, A A, 0.33 l0 l0 l0 10 33 Penicillin 10 33 50 Erythromycin100 100 25 100 or corresponding iron-free compound Table 2: Doses (inmg/kg) of different antibiotics which effect a 75-100% cure whenadministered once.

in vivo. Mice were infected with virulent strains of Staphylococcusaureus, Streptococcus haemolyticus and Diplococcus pneumoniae Type IIIwhich is chemotherapeutically difiicult 'to attack. As infection doseapproximately 100 lethal doses were administered intraperitoneally ineach case; in the untreated control animals this led in 100% to sepsisfollowed by death within 24-48 hours. Treatment was carried out a. fivetimes within hours (first administration k hour before infection, then3, 5, 21 and 30 hours after infection), b. with single doses which weregiven after infection. 7 In both series of experiments the preparationwas administered both subcutaneously and per os. In general, at least 10animals were used in each group. The result of the treatment was checkedon the'tenth day after infection and expressed as a percentage per groupof the surviving animals. The results are put together in Tables l and2.

In experiments on at least ten animals per group the immediatelydifference between a treatment effect of -100% su rvival and a controlgroup mortality of (no survivors) fulfils the requirements of the 2 X 2test for a significance of P 0,01 (Mainland & Murray, 1952, Science1161591-594). Thus, in both series of the above experiments,statistically significant data were obtained at the 1% level (P z 0.01).

The toxicity Ifinarea myans and desferrimycin's'is low. Thus, forexample, mice tolerated the subcutaneous administration of 1000 mg orthe oral administration of 3,000 mg per kilogram of body weight withoutsuffering harm. Higher doses have not been tested.

Because of their good antibacterial activity and their low toxicityferrimycin A, ferrimycin A and ferrimycin A can be used as medicamentsagainst infections caused by the above-mentioned microorganisms,especially infections caused by Staphylococci, Streptococci orPheumococci. The compounds can be applied orally or parenterally, forinstance subcutaneously or intramuscularly. Antibiotic ferrimycin A, itscomponents ferrimycin A, and and ferrimycin A its salts and derivativesare useful as medicaments, for example, in the form of pharmaceuticalpreparations. They can also be used as additives for animal feedstuffsor as disinfectants or preserving agents, The pharmaceuticalpreparations contain the active compounds in admixture with apharmaceutical organic or inorganic carrier suitable for enteral,parenteral or local administration. For making the carrier there areused substances which do not react with the new compounds, for example,gelatine, lactose,starches, magnesium stearate, talc, vegetable oils,benzyl alcohols, gums, polyalkylene glycols, white petroleum jelly,cholesterol or other known carrier for medicaments. The pharmaceuticalpreparations per volume of solution per minute. After incubating for i3may be, for example, in the form of tablets, dragees, powders, salves,creams, suppositories or in liquid form as solutions, suspensions oremulsions. If desired, they may be sterilized and/or may containauxiliary substances, such as preserving, stabilizing, wetting oremulsifying agents. They may also contain other therapeutically valuablesubstances.

FlG. II shows the IR-spectrum in potassium bromide of crude ferrimycin Amonohydrochloride;

FIG. 2 shows the lR-spectrum in potassium bromide of ferrimycin Adihydrochloride;

FIG. 3 shows the IR-spectrum in potassium bromide of ferrimycin Adihydrochloride;

FIG. i shows paper chromatography of various enriched ferrimycinproducts (1 product of Ex. 7; 2 product of Example 10; 3 product ofExample 13; 4 ferrimycin A 5 ferrimycin A in the systems Ibutanol-glacial acetic acid-water 4:1 :5; 10 hours II butanoI-butylacetate-glacial acetic acid-waterlid By using instead of the abovenutrient solution, solutions which contain, per liter of tap water, thefollowing nutrient substances culture filtrates of similarly highantibiotic activity are obtained by incubation and working up in ananalogous manner.

a) Glucose 10 grams Soya bean meal 10 grams Sodium chloride 5 gramsSodium nitrate 1 gram b) Glycerine grams Soya bean meal 10 grams Sodiumchloride 5 grams Sodium nitrate 1 gram Calcium carbonate 10 grams c)Glucose 10 grams Soya bean meal 10 grams Corn steep liquor 20 gramsSodium chloride 5 grams Sodium nitrate I gram Calcium carbonate 10 gramsd) Lactose 20 grams Distillers solubles 20 grams Sodium chloride 5 gramsSodium nitrate I gram 1001131143; 24 hours III butanol-butylacetate-glacial acetic acid-water 100:301131143; 60 hours; FIG. 5 showsthe UV-spectra (in water, c 5.10 of 25 ferrimycin A (Curve 1, ExtinctionE A) ferrimycin A (Curve 2, Extinction E A0. I )and ferrimycin A (Curve3, Extinction E A-0.3); FIG. 6 shows the countercurrent distribution ofcrude ferrimycin'(containing ferrimycin A and ferrimycin B) over 115stages according to Craig. Extinctions at 425 my. (iron colorfcurve I)and at 570 mu (ninhydrin color, curve 2) and in vitro activity (againstStaph. aureus, curve 3) are indicated;

FIG. 7 shows the llR-spectrum in liquid petrolatum of desferrimycin A IFIG. 0 shows the NMR-spectrum in trifluoracetic acid of desferrimycin AThe following examples illustrate the invention:

or. shaking well at 27C, the cultures in the fermenters being aeratedwith about 2 parts by volume of sterile air 48-120 hours the culturesolution has a high inhibiting value against test organisms (B.subtilis, B.megatherium, micrococcus pyogenes, var.aureus). The cultureis interrupted and the pH value is adjusted to 4.5 60 by the addition ofdilute sulfuric acid, and the mycelium and'any other solid material isseparated from the main body of the solution containing the antibioticby filtration or centrifuging, 1% of a filtration assistant, forexample, Hyflo Supercel, being added if desired to 5 the culturesolutionbefore filtration. The filter residue is washed with water and withaqueous methanol, and the washings are united with the culture filtrate.

EXAMPLE 2 3 Liters of a culture filtrate obtained as described inExample I, are adjusted to a pH value of 7.5 by the addition of a dilutesolution of caustic soda, and gramstered, the carbon residue beingextracted once more in the same manner. The eluate contains the wholeantibiotic activity.

EXAMPLE 3 3 Liters of a culture filtrate obtained as described inExample I are adjusted to a pH value of 7.5 by the addition of a dilutesolution of caustic soda, and then the solution is immediatelysuppliedat the rate of 0.5 liter 0 per hour to a column of AmberliteIRC-SO (I-I-form) having a length of 30 centimeters and a diameter. of 5centimeters. The antibiotic is completely adsorbed by the column. Thecolumn is washed withfi3 liters ofwater. For the purpose of elutionthere is used one liter of 5 0.4N-hydrochloric acid, the first 500 cc ofthe'eluate are antibiotically inactive, whereas the second 500 cccontain the whole activity. In order to remove theexcess of hydrochloricacid this active eluate is percolated through a column of AmberliteIR-4B (a weakly basic polystyrene resin containing polyamine exchangegroups). By freeze drying the percolate' the enriched antibioticferrimycin is obtained in the form of ahighly active amorphous powder.

. EXAMPLE 4 6 Liters of a culture filtrate obtained as described inExample 1 are adjustedto a pH of 7.5 by the addition of dilute causticsoda solution, and then immediately percolated through a column of Asmit173, 14 cm long and having a diameter of 4.5 centimeters. The antibioticis completely adsorbed. The adsorbant resin is then washed with 3 litersof water and the antibiotic is eluted, with 1 liter of a mixture ofmethanol and 1N- acetic acid lzl The eluate, which contains the whole ofthe active substance, is concentrated in vacuo at 35C to 15 cc. 75 cc ofa 0.25N-solution of hydrogen chloride in methanol is then added to thesolution, and the mixture is poured into 10 liters of acetone, wherebythe hydrochloride of the antibiotic is precipitated. The antibiotic isfiltered off and washed with acetone. For the purpose of furtherpurification it is dissolved in 150 cc of methanol, and the somewhatturbid solution is filtered with the addition of Celite (an infusorialearth). By evaporating the filtrate in vacuo at 25C, the hydrochlorideof antibiotic ferrimycin is obtained in the form of an amorphous powder.

EXAMPLE 5 30 Liters of a culture filtrate obtained as described inExample 1 are adjusted to a pH value of 4.5, and then concentrated to 2liters in a thin-layer evaporator. The concentrate is adjusted to a pHvalue of 8 by the addition of dilute caustic soda solution, and themixture is then filtered with the addition of Celite. The clear filtrateis adjusted to a pH value of 5, and 1.5 liters of a hot aqueous solutionof picric acid of 5 percent strength are added while stirring. Theprecipitate so formed, after being allowed to stand for several hours isfiltered off at C with the addition of 50 grams of Celite. The filtratehas only a very slight antibiotic activity. The filter residue is thenstirred three times with 800 cc of cold acetone each time and filtered.The filtr, ate is concentrated in vacuo to 80 cc, whereupon the picrateof the antibiotic and excess of picric acid precipitateout. Afterseparation there are obtained 8.5 grams of dry substance.

In order to isolate the antibiotic in the form of its hydrochloride 2grams of the aforesaid dry substance are dissolved in 30 cc of methanol.There are then added first a mixture of 1 cc of concentratedhydrochloric acid and cc of acetone, and subsequently 500 cc of ether,whereupon'the hydrochloride precipitates out quantitatively. By repeateddissolution of the precipitate in methanol acidified withhydrochloricacid and precipitation with ether the last residues of picric acid areremoved. Finally, the hydrochloride is dissolved in as small a quantityas possible of methanol, andthe solution is filtered and evaporated invacuo. There are obtained 714 mg of the hydrochloride of antibioticferrimycin.

EXAMPLE 6 .600 liters of a culture filtrate obtained as described inExample 1 are stirred with 5.5 kilograms of Hyflo .Supercel, adjusted toa pH value of 4 with 2.5 liters of 2N-HCl and then filtered. The filterresidue is washed with 100 liters of water. The clear filtrate isstirred with 7 kilograms of pretreated Norit for 45 minutes. Thepretreatment of the Norit is carried out by stirring several times withlN-HCl and then washing adsorbate twice for one hour with a mixture ofnbutanol-methanol-glacial acetic acid and water (2:1: 1:2), the activecharcoal being separated by filtration. The combined eluates (140 liters46 liters) are mixed well with 96 liters of butyl acetate. The aqueousphase is separated and the organic phase is washed with 1.2 liters ofwater. The combined aqueous phase (65.5

liters) are washed in succession by stirring with 72 liters EXAMPLE 7For the purpose of isolating antibiotic ferrimycin from 300 liters of aculture filtrate obtained as described in Example 1, 1 part by volume ofAmberlite [RC 50 in the Hform is mixed mechanically with 2 parts byvolume of Amberlite IRC 50 in the Na-for m. 6.3 liters of this mixtureare poured into a glass column. The ratio of the height to diameter ofthe resin filing is 8:1. The culture filtrate is adjusted to pH 4 with2N HCl and percolated through the resin at a rate of flow of 0.2

liter per minute per literof resin, an orange-brown zone being formed inthe upper two-thirds of the column. The resin is then washed with 30liters of water and with 60 liters of methanol of 80% strength. Therunnings and the washings contain only little antibiotic activity.Elution is carried out in two portions with a total amount of 37 litersof a mixture of 8 parts by volume of methanol and 2 parts by volume of1N HCl. Both portions are adjusted to pH 5 with 5N NaOH, combined andthen concentrated to 2 liters at a temperature of at the most 30C in acirculation evaporator. The aqueous concentrate is adjusted to pH 5.6and filtered through Hyflo Supercel for the purpose of removing anyinsoluble material. The filtrate (2.3 liters) contains approximately thewhole antibiotic activity of the chloroform and extracted three timeswith 500 ml of 1/10 N-ammonium acetate buffer of pH 4.60 each time,colored antibiotically inactive impurities thus being removed from theorganic phase. The chloroform phase is then extracted first with 300 mland then twice with ml of l/ ION HCl each time. The deep red coloredacid extract containing the antibiotic is adjusted to pH 3.5 withpotassium bicarbonate and re-extracted with four 50 ml portions of aphenol-chloroform mixture (100 grams 100 ml). The phenol-chloroformextract is filtered through Celite. To the clear, redcolored filtrate(200 ml) 50 ml of water,'500 ml of ether and 300 ml of petroleum etherare added with vigorous agitation. After separating the aqueous phase,

the organic phase is washed twice with 50 ml of water each time. Thecombined aqueous extracts are extracted twice with 500 ml of ether eachtime and once with 500 ml of benzene and then lyophilized. There areobtained 2.60 grams of an antibiotically highly active orange-browncolored powder. This material shows 5001 ,000 times more specificantibiotic activity compared with the starting material (activity perweight unit of dry substance). Paper-chromatography of this material onWhatman No. l paper in n-butanol: n-butyl acetate: glacial aceticacidrwater (:3:1. 3:143) system reveals two spots after autobiographicdevelopment with Staphylococcus aureus. The slowly travelling antibioticsubstance is designated Base A, the substance travelling 2.5 times morequickly Base B. Base A gives a blue color reaction on paper on beingsprayed with ferric chloride and potassium ferricyanide.

EXAMPLE 8 338 Grams of an antibiotic preparation obtained as describedin Example 6 are dissolved in 1.5 liters of water. 150 Grams ofcrystalline ammonium sulfate are added and the solution is extractedfirst with 1 liter and then twice with 500 ml each time of aphenolchloroform mixture containing 100 grams of phenol in 100 ml ofchloroform. The combined phenol chloroform extracts are extracted with750 ml of in hydrochloric acid and then filtered through a layer ofCelite. To the clear red-brown filtrate there are added 600 ml of water,4 liters of ether and finally 4 liters of petroleum ether with stirring.The aqueous phase is separated and the organic phase extracted with 200ml of water twice. The combined aqueous phases l liter) are washed twicewith 1 liter of ether and then lyophilized, 136 grams of a brown powderare obtained which has a specific antibiotic activity twice as high asthe starting material.

EXAMPLE 9 550 mg of highly active antibiotic preparation (Base A)obtained as described in Example 11 are dissolved in 55 ml of a 1/ION-ammonium acetate buffer having a PH value of 4.6 and extracted 4times with 20 ml of a phenol-chloroform mixture containing '100 grams ofphenol in 400 ml of chloroform. The organic extracts are re-washed twicewith 15 ml buffer solution. The organic extract (80 ml) containing theantibiotic is diluted with 40 ml of chloroform, washed once again with60 ml of buffer solution and then filtered through a double pleatedfilter. The deep red-colored filtrate is extracted successively with 30,20, and 10 ml of 0.2N- hydrochloric acid. The acid solution containingthe antibiotic is diluted with 50 ml of water and exhaustively extractedtwice with and then with 10 ml of a mixture of phenol and chloroformcontaining 100 grams of phenol in 100 ml of chloroform. The combinedphenolchloroform extracts are filtered through a double pleated filterand agitated with 20 ml of water, 200 ml of ether and 100 ml ofpetroleum ether. After separating the aqueous phase, the organic phaseis reextracted twice with 15 ml of water. The combined aqueous extractsare washed twice with 50 ml of ether and once with 50 ml of benzene andthen lyophilized. There are obtained 1 17 mg of a brown-red powder whichhas approximately five times the amount of antibiotic activity comparedwith the starting material.

EXAMPLE 10 v 700 mg of the antibiotic preparation obtained as describedin Example 7 are chromatographed over 127 grams of Whatman No. 1cellulose powder. For the purpose of elution there is used the upperphase of a mixture of 4 parts of butanol, 1 part of glacial acetic acidand 5 parts of water, to which are added 10% by volume of butanol. Thesubstance is triturated with ten times the quantity of cellulose powderand put on the column as powder. The rate of running through is 15-20 mlper hour. Fractions of 40 ml are collected. The separate fractions areagitated with 50 ml of petroleum ether. The separated aqueous phase iswashed with benzene and lyophilized. The major portion of the antibioticactivity is in the fractions 7-8 (121 mg) and l0-l3 (142 mg). On beingexamined by paperchromatography it is found that the fractions 7-8contain chiefly Base B and fractions 110-13 primarily Base A.

EXAMPLE 1 1 3 Grams of an antibiotic preparation obtained as describedin Example 8 are distributed over a hundred stages in a Craigsdistributing apparatus in the secondary butanol 0.lN-ammonium acetatebuffer system having a pH value of 4.68, each unit contains ml upperphase and 100 ml lower phase and the substance being charged into unitNo. 3. After distribution the content of each unit is worked up byadding double the volume of petroleum ether to the mixture of the twophases and freeze-drying the aqueous phase; The dark- .colored fractions3-ll show only little antibiotic activity. The orange-yellow coloredfractions l2-20 (631 mg) contain the major portion of the antibioticactivity (chiefly Base A). The yellow-colored fractions 21-40 are lessactive and contain a mixture of Base A and Base B in which the latterpredominates.

EXAMPLE 12 220 mg of an antibiotic preparation (Base A) obtained asdescribed in Example 10' are distributed over 29 stages in a Craigsdistributing apparatus in the 1 1ONammonium acetate buffer (pH value4.58) 10% phenol in chloroform. Each stage contains 10 ml each of upperand lower phase. The major portion of the antibiotic activity is infractions 6 15. The latter ,are combined (about 200 ml), 400' ml ofether and-300 ml of petroleum ether are added and the whole agitated.The separated, orange-red colored aqueous phase is washed withchloroform and extracted successively with 20 and three times with 10 mlof a'mixture of 100 grams of phenol in 100 ml of chloroform; 20 ml ofwater, 300 ml of ether and 200 ml of petroleum ether are added to thephenol-chloroform extracts with agitation. The red-colored aqueous phaseis separated, washed with much ether and-benzene and lyophilized. Thereare obtained 80.4 mg of Base A in the form of a yellow water-solublepowder. Color reactions: FeCl brown-red; FeCl K Fe(;CN) blue;

Ninhydrin: weakly positive.

Negative: Sakaguchi, Maltol, Elson-Morgan.

EXAMPLE l3 8 grams portions of an enriched ferrimycin product (maincomponent A: activity in relation to the lyophilized culture filtrate2,000 to 3,000) such as is ob tained for example, according to Example10 were subjected in a vertical glass column having a length of 1 meterand a diamter of 6 centimeters which was filled with cellulose powderand provided with a cooling jacket, to zonal electrophoresis by themethod of J. Porath [Biochimica et Biophysica Acta, Vol. 22 page 151(1956)]. As an electrolyte solution there was used 54; N-acetic acid.The product was dissolved in 160 ml of water, and the brown-red solutionwas poured on to the upper anode end of the column. At a voltage of1,000 and a current of 100 milliamperes the orange antibiotic zone about20 cm wide migrated towards the cathode at a speed of 3.3 cm per hour.In order to increase the separating action of the column this electricmovement was exactly compensated by a stream of the electrolyte movingthe opposite direction at the rate of 81 ml per hour, and in this waythe antibiotic was held stationary in the same place in the column.Brownish colored inactive accompanying substances having higher or lowerelectrical migration speeds than that of the antibiotic were moved tothe cathode zone or anode zone, and wahsed away from the zone eithercontinuously or periodically. After a period of electrophoresis lasting-6 days the antibiotic had moved through a liquid column 4-5 meterslong. The substance on the column was then eluted with electrolytesolution, and the eluate fractions collected in an automatic fractionreceiver were tested biologically. The deep. red colored biologicallyactive fractions were combined (1.3 liters). From the acetic acidsolution the antibiotic could be extracted after the addition of 75 mlof a saturated aqueous solution of sodium chloride, with 250 ml of amixture of phenol and chloroform (lgzlml). After filtering the extractthrough Celite, the antiobiotic could be precipitated as an orangecolored precipitate on 16 grams of Hyflo Supercel by the addition of 1liter of ether and 500 ml of petroleum ether. The mixture of theprecipitate and filtration assistant was washed well with acetone andfinally extracted with a small amount of cold methanol. From themethanol extract the crude ferrimycin A monohydrochloride wasprecipitated with acetone in the form of an orangecolored powder. Afterbeing dried at room temperature under 0.001 mm pressure for two days theproduct contained approximately 100% of the original activity in a formenriched four to six times. Analysis: C 50.68%; H 6.99%; N =.13.45%; Fe(gravimetric) 3.55%; Fe (colorimetric) 3.66%; Cl =2.75%; 2.46% dryingloss at 120C under 0.01 mm pressure.

Infra-red spectrum in potassium bromide; see FlGl. It shows bands at2.97; 3,43; 6.05; 6.30; 6.92; 7.25; 7.98; 8.25; 8.40; 8.95; 13.20 pt.

Ultra-violet-spectrum in water; maximum at 318 mp. (E 47.2); inflexionsat 229 mp. and at 400 mu. Solubility: dissolves very well in water,methanol and mixtures of phenol and chloroform, soluble indimethylformamide, methyl-cellosolve, benzyl alcohol and glacial aceticacid, sparingly soluble in ethanol and practically insoluble inpyridine, propanol, butanol and the usual organic solvents, especiallylipoid solvents.

Precipitation reactions: precipitatable from aqueous solution by meansof picric acid, picrolonic acid, and ammonium reineckate.

Color reactions: orange-red aqueous solutions change color upon theaddition of mineral acid reversibly through claret red to pale yellow.Decoloration is likewise caused by caustic soda solution. Neutralsolutions do not react with potassium ferrocyanide. Acidified solutionsgive colorations or precipitations of Prussian blue. Trivalent ironbound in complex union is liberated as the pH-value decreases.

Ferric chloride produces a claret red coloration and a mixture of ironchloride and potassium ferricyanide gives a blue coloration. Thefollowing tests are negative: Molish, Anthron, Folin Ciocalteu,Sakaguchi. The ninhydrin reaction in butanol-pyridine is weakly positiveafter heating for a long time. Hydrolysis with 6N- hydrochloric acidyields a mixture of about 15 substances detectable by paperchromatography.

lron-free ferrimycin A:

mg of the purified ferrimycin A so obtained were dissolved in 1 ml ofmethanol. 334 mg of ooxyquinoline in 2ml of methanol were added and thewhole was allowed to stand at room temperature for 8 hours. Afterallowing the whole to stand for a further 15 hours at 0C theprecipitated black-green crystals of iron 8-oxyquinoline were separated.The solution was diluted with a small amount of water and extracted bythorough agitation with chloroform and benzene to remove the excess ofoxyquinoline. The remaining pale yellow aqueous phase, which containsthe iron-free antibiotic was lyophilised: 90 mg of a beige powder. Theiron-free antibiotic exhibited the same absorption spectrum in theultraviolet region as the starting material, but in the visible regionit lacked the flat band at 400-430 mu. It had only /i of the activity invitro of the starting material. A colorless solution of the iron-freeantibiotic is instantaneously colored deep red on the addition of ferricchloride, whereupon the iron absorption at 400-430 my. in the spectrumreturns and the specific antibiotic activity is increased.

EXAMPLE 14 800 mg of a preparation of ferrimycin A obtained as describedin Example 13 were chromatographed over a cellulose-column measuring 3 X65 cm (198 grams) at 12C. As flowing agent there was used the systemtertiary butanol/0.001N hydrochloric acid/saturated aqueousNaCl-solution (2:1 :1 The fractions collected in an automaticfractionreceiver amounted to 30-40 ml and were examined. biologically,spectroscopically and by paper chromatography. The fractions 60-104contained 191 mg of ferrimycin A fractions -200 contained 92 mg offerrimycin A The correspondingly unified fractions were agitatedwith anequal volume of petroleum ether and with about 10% by volume of water,whereby the antibiotic substance was driven into the aqueous phase. Fromthe latter it was worked up by the method described in' Example 13 witha mixture of phenol and chloroform and was obtained in the form of anorange powder. After being dried at 25C under 0.001 mm pressure for 50hours over P O /KQH the product had the following properties: FerrimycinA dihydrochloride: Analysis (average from three determinations): C46.71%; H 6.80%; Fe 5.33%; N

12.70%; C1 6.76%; O (calc.) 21.70%. Titration in 80% methylcellosolve;equivalent weight: 1078; pK.

4.11; 7.92 and 11.4. Ultra-violet spectrumzltmax 229' mp.(E 336), 319my. (E 37) and 425 mp. (E 27.6). Infra-red spectrum potassium bromide:see FIG. 2. It shows bands at 3.00; 3.44; 3.51; 6.08; 6.31; 6.90; 7.10;7.35; 7.45; 7.95; 8.41; 9.00; 10.25; 11.85; 13.20 1L.

Ferrimycin A -dihydrochloride: Analysis: C 45.78%; H 6.77%; N 12.75%; C16.23%; Fe 5.29%; titration in 80% methyl-cellosolve; molecular weight:1086; pK: 4.04, 7.91 and 11.05. Ultra-violet spectrumzA max. 227 mp. (E332), 319 mp. (E 37) and 425 mp. (E 25). Infra-red spectrum (inpotassium bromide): see FIG. 3. The paper chromatography of variousenriched and purified ferrimycin products on Whatman No. l-paper isshown in FIG. 4. The test was made bio-autographically withStaphylococcus aureus.

In FIG. 4 the symbols have the meanings: 1: System butanol glacialacetic acid-water 411:5; 10

hours 11: System butanol-butyl acetate-glacial acetic acidwater100:30z13z143; 24 hours 111: System butanol-butyl acetate-glacial aceticacidwater 100:30:l3:l43; 60 hours. 1: Antibiotic ferrimycin, base A-l-B,according to Example 7 (1 pg) 25 Antibiotic ferrimycin B, according toExample 1 Mg) 3: Antibiotic ferrimycin A according to Example 13 (0.1pg) 4: Antibiotic ferrimycin Al, according to this Example (0.05 ,ug) 5:Antibiotic ferrimycin A2, according to this Example (0.05 ug).

EXAMPLE l5 6 grams of ferrimycin product having about 1,000 times theantibiotic activity of the lyophilized culture filtrate and containingferrimycin A as well as ferrimycin B, are distributed by countercurrentover 115 stages according to Craig. The apparatus consists of 120 units.It is filled per unit with 100 cc of upper phase and 100 cc of lowerphase of a mixture, equilibrium at 19C, of benzyl alcohol (200 parts byvolume), nbutanol (100 parts by volume), N-hydrochloric acid (6 parts byvolume), water (300 parts by volume) and aqueous sodium chloridesolution (60 parts by volume) saturated at 19C. The first two unitsmerely contain solvent. In each of the following three units 2 grams ofsubstance are introduced and the whole distributed 1 15 times at 19C.The number of shakes per distribution is 30, the duration of intervals15 minutes.

When the distribution is complete, the resulting 118 fractions are keptat C. From every third fraction there are taken for test purposes 10 ccof the upper and 10 cc of the lower phase which are agitated with 50 ccof petroleum ether. The separated aqueous phase (10 cc) which nowcontains all the hydrophilic material is freed from any adhering organicsolvent by brief evacuation. The resulting test solutions are used onthe one hand for colorimetric evaluation (extinction at 425 my. in 1 cccuvette. Compare FIG. 6, curve 1) and for the ninhydrin color reaction.To carryout the latter 0.5 cc of test solution and 0.5 cc of ninhydrinreagent, prepared is described by S. Moore and W. H. Stein, J. Biol.Chem. 211, 907 (1954), are mixed, heated at 100 C for minutes, dilutedwith 5 cc of a mixture of alcohol and water (1:1) and then measured in aspectrophotometer at 570 mu (curve 2). For biological tests the testsolutions are diluted 1:50. In curve 3 there are shwon the in vitroactivities against Staphylococcus aureus in the plate diffusion test inrelation to an arbitrary standard. The active fractions 40-70 contain,as can be shown by paper chromatography, ferrimycin A, whilst ferrimycinB is in fractions 75-100. A considerable enrichrnent can be achieved.The antibiotically active fractions 25-39, 40-48, 49-55, 56-70 and 71-95are combined and agitated with the same volume of petroleum ether.Thered colored substancesare driven into Fraction Quantity EnrichmentYield of Ferrimycin in mg factor activity The starting material isobtained as follows:

30 liters of an aqueous eluate concentrate obtained as described inExample 7 by elution of ferrimycin from Amberlite IRC 50 and subsequentremoval of the methanol in vacuo, are mixed with 1 kg of Hyflo withstirring and then in the course of 2 hours with 1.8 liters of a mixtureof 11 parts of phenol and 1 part of water. Stirring is then continuedfor half an hour, and the mixture is then suction-filtered. The filtratecontains about 2-5 percent of the activity and is discarded. The wellsqueezed filter cake is stirred twice with 4 liters of ether each timeand once with a mixture of 8 liters of acetone and 2 liters ofv etherand suction-filtered on each occasion, whereupon the residue is washedwith succession on the filter with 5 liters of acetone and 4 liters ofchloroform. All the washings are practically inactive. Thechloroform-moist filter-cake is then introduced into 3 liters of amixture of phenol and chloroform 1:1

(weight/volume) and stirred for 1 hour. In the course.

of 1 hour 15 liters of chloroform are added in a uniform current. TheHyflo is suction-filtered and washed twice with 2 liters ofphenol-chloroform 1:11 (weightlvolu me) and once with 2 liters oichloroform. The residue contains 5-10% of the activity.

The combined active filtrate (22 liters) are concentrated to 4 liters at25C and the concentrate added dropwise in the course of 30 minutes to amixture of 4 liters of ether, 8 liters of petroleum ether and 400 gramsof Hyflo. After another 30 minutes the mixture is suction-filtered. Thefilter-cake is washed with 2 liters of ether and twice with 1 liter ofacetone. All the filtrates contain only traces of activity.

The filter-cake is stirred three times for 10 minutes with 1.5 liters ofmethanol each time, suction-filtered and finally washed with 0.5 literof methanol onthe filter. The washed Hyflo does not contain anyactivity.

The dark brown filtrates (4 4.5 liters) are evaporated to drynesscautiously at 20-30C in a water-jet vacuum. The still sticky residue isdried in a high vacuum for 20 hours. 75-88 grams of a red-brown stronglyactive ferrimycin product are obtained. The yield of activity calculatedon the starting material is EXAMPLE 16 2 grams of a ferrimycin A product(fractions 49-55) obtained as described in Example 115 which has about9,000 times the antibiotic activity of the lyophilized culture filtrate,are chromatographed on a 70 cm X 7.14 cm column of a strongly acid ionexchanger Dowex 50 WX (/200 mesh). The ion exchanger is first purifiedaccording to Hirs et: al., J.Bi0l.Chem 219,

1. FERRIMYCIN A1 OF THE FORMULA C41H65O14N10FE HAVING PKMCS VALUES OF4.11, 7.87 AND 11.4 AND EXHIBITING THE FOLLOWING MAXIMUM IN THEULTRAVIOLET SPECTRUM: $MAX 229 MU(E1CM 1% =336), 319 MU(E1CM 1%=37) AND425 MU(E1CM 1% =27.6) AND THE IR-SPECTRUM IN POTASSIUM BROMIDE SHOWN INFIG.
 2. 2. Ferrimycin A2 of the formula C41H68O14N10Fe having pKMCSvalues of 4.04, 7.91 and 11.05 and exhibiting in the ultravioletspectrum the following maxima: lambda max 227 m Mu ( Alpha 1cm1% 332),319 m Mu ( epsilon 1cm1% 37) and 425 m Mu ( epsilon 1cm1% 25) and theIR-spectrum in potassium bromide in FIG.
 3. 3. Desferrimycin A1 havingthe empirical formula C41H68O14N10, having pKMCS values of 4.06 and 7.79and ( Alpha )D20 -25* (in ethanol) and exhibiting in the ultravioletspectrum the folloiwng maxima: 210 m Mu (log epsilon 4.57), 233 m Mu(log epsilon 4.38) and 322 m Mu (log epsilon 3.22) and having theIR-spectrum in liquid petrolatum shown in FIG. 7 and the NMR-spectrum intrifluoracetic acid shown in FIG.
 8. 4. A process for producingferrimycin A, which comprises cultivating Streptomyces griseoflavus NRRL2717 in an aqueous nutrient medium containing a source of carbon andnitrogen and inorganic salts under aerobic conditions at 18*-40*C untilthe nutrient medium exhibits a substantial antibacterial activity, andthen isolating the ferrimycin A from the culture filtrate.